Transcriptome analysis reveals genes associated with late blight resistance in potato

Late blight is a serious disease of potato worldwide. Our study aimed to unveil genes involved in late blight resistance in potato by RNA-seq analysis after artificial inoculation under controlled conditions. In this study, two potato somatic hybrids (P7 and Crd6) and three varieties such as Kufri Girdhari, Kufri Jyoti and Kufri Bahar (control) were used. Transcriptiome analysis revealed statistically significant (p < 0.05) differentially expressed genes (DEGs), which were analysed into up-regulated and down-regulated genes. Further, DEGs were functionally characterized by the Gene Ontology annotations and the Kyoto Encyclopedia of Genes and Genomes pathways. Overall, some of the up-regulated genes in resistant genotypes were disease resistance proteins such as CC-NBS-LRR resistance protein, ankyrin repeat family protein, cytochrome P450, leucine-rich repeat family protein/protein kinase family, and MYB transcription factor. Sequence diversity analysis based on 38 peptide sequences representing 18 genes showed distinct variation and the presence of three motifs in 15 amino acid sequences. Selected genes were also validated by real-time quantitative polymerase chain reaction analysis. Interestingly, gene expression markers were developed for late blight resistant genotypes. Our study elucidates genes involved in imparting late blight resistance in potato, which will be beneficial for its management strategies in the future.

Late blight, caused by the oomycetes Phytophthora infestans, is the most serious disease of potato.This pathogen is a highly variable and severely damages potato crops, and therefore its management is a challenging task.During the 1960s-1980s, race-specific resistance genes were deployed in potato breeding using the hexaploid wild species Solanum demissum 1 .However, over the decades, the R genes were defeated due to the emergence of new P. infestans strains.Hence, there is a need to identify new resistance sources in wild species background, of which many of them are yet to be characterized at transcriptome level.
The genus Solanum is a rich source of genetic diversity, containing over 200 wild species 2 .Several wild species have been identified in potato, which confer late blight resistance such as S. pinnatisectum, S. cardiophyllum, S. bulbocastanum, S. stoloniferum, S. bulbocastanum, S. demissum, S. polytrichon, and S. microdontum 3 .Many useful genes derived from wild species cannot be transferred through conventional breeding due to sexual barriers caused by differences in the ploidy number and the endosperm balance number 4 .Hence, somatic hybridization has been deployed via protoplast fusion to overcome crossing barriers 5 .For example, we developed interspecific potato somatic hybrids through protiplast fusion for late blight resistance namely P7 (Solanum tuberosum + S. pinnatisectum) 6 , and Crd6 (S. tuberosum + S. cardiophyllum) 7 .However, these somatic hybrids have not yet characterized at transcriptome level for late blight resistance genes.
With the advancement in the post-genomics era and availability of the potato genome 8 , it is now feasible to analyse genes at the whole genome level in potato.Many reports are available on whole transcriptome sequencing for late blight resistance in potato [9][10][11] .Transcriptomics studies have been performed on host-pathogen interaction to discover gene networks regulating late blight resistance in potato 12 .In addition, many candidate genes have been identified in potato for multiple traits such as late blight, potato virus Y and bacterial wilt causing pathogens interaction 13 , wild species S. pinnatisectum conferring late blight resistance 14 , contrasting potato foliage and tuber defense response 15 , and scion grafted to potato rootstock for improving late blight resistance 16 .
In this study, we aimed to identify genes associated with late blight resistance by transcriptome sequencing in interspecific potato somatic hybrids and varieties after artificial inoculation.Differentially expressed genes

Venn diagram analysis and gene expression marker development
Venn diagram analysis showed common genes in four genotypes such as P7, Crd6, Kufri Girdhari and Kufri Jyoti (Fig. 4).In which, P7 and Crd6 shared only 39 common up-regulated genes and 65 down-regulated genes.

GO annotation and KEGG pathways analysis
All DEGs were functionally characterized by GO terms where molecular function showed the highest gene counts (65,645) followed by biological process (54,476) and cellular component (48,301) (Supplementary Table S2).
The WEGO plots are depicted in Fig. 5 for up-regulated and down-regulated genes in P7 and Kufri Girdhari (Supplementary Excel datasets # 5-8).The GO annotations of genes in CRd6 and Kufri Jyoti are shown in Supplementary Fig. S1.Overall, a few GO terms were found predominantly such as catalytic activity, binding, metabolic process, cellular process, cell, and membrane.All DEGs were classified into 24 KEGG functional pathways categories, which included KEGG annotated gene counts such as P7 (5448), Crd6 (5428), Kufri Jyoti (5414) and Kufri Girdhari (5478) (Fig. 6) (Supplementary Tables S3 and S4) and detailed in Supplementary Excel datasets (#9-12).In all combinations, maximum KEGG annotated gene counts were found for signal transduction than other pathways like translation, carbohydrate metabolism, folding sorting and degradation, amino acid metabolism, energy metabolism, lipid metabolism and transport, catabolism, cell growth and death, and environmental adaptation.Scatter plot and volcano plot analysis showed up-regulated and down-regulated genes are shown in Supplementary Figs.S2 and S3.

Phylogeny tree and conserved motif analysis in selected genes
Multiple sequence alignments of 38 amino acid sequences belonging to 18 selected genes were performed (Supplementary Table S5).These selected genes belonged to disease resistance like bacterial spot disease resistance protein 4, disease resistance proteins, disease resistance protein RPM1, late blight resistance protein homolog R1B-23, CC-NBS-LRR resistance proteins, LRR family proteins, NBS-LRR resistance proteins, cytochrome P450, MYB and AP2-EREBP TFs.Subsequently, the aligned amino acid sequences were used for phylogeny analysis using the MEGA software.The Neighbor-Joining tree was constructed, which clearly distinguished the 38 amino acid sequences representing 18 genes into five major clusters (I-V) (Fig. 7).Cluster I contained a total of  (2).On the other hand, cluster III contained 6 sequences such as CC-NBS-LRR resistance protein (1), resistance gene (1), bacterial spot disease resistance protein 4 (4).Interestingly, cluster IV has only one sequence of disease resistance protein RPM1 (DMG400029405; DMP400051212).Lastly, cluter V possessed total 13 amino acid sequences including late blight resistance protein homolog R1B-23 (3), disease resistance proteins (10).Thus, phylogeny anaysis indicated genetic relationship among the selected resistance genes, TFs and CC-NBS-LRR/NBS-LRR/LRR proteins conferring late blight resistance in potato genotypes.Furthermore, these amino acid sequences were scanned using InterProScan database, and observed common family/domain particularly disease resistance protein, plants (IPR044974) in all resistance genes and CC-NBS-LRR proteins.Whereas, other TF revealed different domains like MYB domain (IPR017930), LRR domain (IPR032675) and AP2/ERF domain (IPR044808).Sequences were also analysed and three conserved motifs alongwith motif locations were predicted using the MEME software (Fig. 8).

Validation of selected genes by RT-qPCR analysis
Selected eight genes (2 genes from each sample of both up-regulated and down-regulated) were validated by RT-qPCR analysis using Kufri Bahar as highly susceptible control.RT-qPCR analysis was performed for one of each gene from up-regulated and down-regulated DEGs, such as disease resistance protein and C protein for P7; gamma aminobutyrate transaminase isoform 2, and AP2/ERF domain-containing transcription factor for Crd6; bacterial spot disease resistance protein 4 and leucine-rich repeat protein in Kufri Girdhari; and disease resistance protein and protein kinase domain-containing protein in Kufri Jyoti.RT-qPCR results are consistent with the RNA-seq results with minor variation in gene expression values (Supplementary Table S6).

Transcriptome profiling for late blight resistance in potato
We provide an overview of global gene expression profiles for genes associated with late blight resistance in potato by total RNA sequencing.This study included highly resistant interspecific potato somatic hybrids P7 (S. pinnatisectum-originated) and Crd6 (S. cardiophyllum-originated) and highly resistant potato variety Kufri Girdhari, and popular but susceptible variety Kufri Jyoti compared with highly suscetible Kufri Bahar (control).Genes were analysed after artificial inoculation of P. infestans at 96 h post-inoculation stage.Our findings are in consistent with previous results on late blight resistance assays in potato on both cultivated and wild species, including our interspecific somatic hybrids 6,7 .Whole transcriptome analysis has identified genes in contrasting resistant and susceptible genotypes 9,10 .Our RNA-seq results were also in accordance with earlier findings on gene identification through microarray technology in potato cv.Kufri Girdhari 17 .This study provides an overview of molecular signatures of somatic hybrids (P7 and Crd6) and varieties (Kufri Jyoti and Kufri Girdhari) suggesting genetic make-up consisting of a network of resistance genes, TFs and stress related genes.Our study sheds light on improving understanding of the induced genes associated with late blight resistance in potatoes.

Disease resistance genes play major role in late blight resistance in potato
Disease resistance proteins play an important role in late blight resistance in potato.Resistance (R) proteins in plants mediate the recognition of specific pathogen-derived factors called avirulence (Avr) proteins.Upon Avr perception, R proteins initiate defence responses that limit further pathogen progress.These responses often    result in macroscopically visible cell death, referred to as the hypersensitive response.To illustrate, highly upregulated genes in resistant genotypes were bacterial spot disease resistance protein 4, CC-NBS-LRR resistance protein, disease resistance protein RPM1, and Sn-2 protein.Earlier, we observed similar group of genes such as disease resistance genes through microarray technology in potato conferring late blight resistance 17,18 .
Plant defense response consists of a multitude of reactions after pathogen infection.The majority of R proteins are NBS-LRR proteins containing a central nucleotide-binding and hydrolysing domain (NB-ARC) and a C-terminal leucine-rich repeat (LRR) domain.Many R genes like putative disease resistance protein over-or under-expressed during host pathogen interaction.It is known that defence-related R gene as well as signal molecules are induced at 72 h post-inoculation, which results in metabolic changes in plants 19 .In this study, 96-h post inoculated samples were analysed due to delay in reaction response between resistant and susceptible genotypes, which might be due to different P. infestns strains, controlled environmental conditions and genotype response.Recently, Duan and co-workers 12 identified higher induction of susceptibility genes such as SWEET after P. infestans inoculation on potato.Our results provide a valuable resource for understanding the interactions between P. infestans and potato at the 96-h post-inoculation stage.
The Indian potato variety Kufri Jyoti was developed in the year 1960s possessing late blight resistance due to the presence of R genes derived from the hexaploid wild species S. demissum.But due to the evolution of new strains of P. infestans, resistance has broken down in this variety and it is susceptible now.Another newly released potato variety Kufri Girdhari was developed in the year 2008, which contains high resistance to late blight.This is might be due to the presence of several resistance genes as observed in this study, such as CC-NBS-LRR type resistance protein (PGSC0003DMG400008596), disease resistance protein RPM1 (PGSC0003DMG400029405), disease resistance protein (PGSC0003DMG400018464), MYB transcription factor MYB139 (PGSC0003DMG402010883) and late blight resistance protein homolog R1B-23 (PGSC0003DMG400025545).Probably these genes provide late blight resistance in Kufri Girdhari against P. infestans infection.

Transcription factors (TFs) regulates gene expression to provide late blight resistance
TFs are one of the key regulators in plant metabolism.TFs constitute an important part of gene networks and signalling pathways in biotic/abiotic stress response.Indeed, the regulation of defense gene expression is largely governed by specific transcription factors.In this study, highly up-regulated TFs genes in resistant genotypes were MYB, MYB139, AP2-EREBP, and C2H2L TFs, thylene-responsive transcription factor 1B and many other genes.Highly down-regulated TFs were C2H2-type zinc finger protein, AP2/ERF, TSRF1, WRKY, R2R3, leucine-rich repeat protein and others.Previous study witnesses that genes with putative functions of transcription-related, such as heat shock protein transcription factor, zinc finger ring-box protein-like and NAC domain-containing protein NAC22, and MYB44 TF are induced after P. infestans inoculation 19 .Our findings are accordance with the previous findings showing that NAC domain protein is a family of plant-specific transcription factors involved in plant development and disease resistance 20 .
The WRKY and MYB TFs have been demonstrated to play key roles in plant responses to stresses, particularly P. infestans infection 21,22 .The zinc finger proteins play a pivotal role in the regulation of plant defense mechanisms against P. infestans infection.Hypersensitivity plays a major role in the induction of disease resistance pathway, and acts as a downstream signalling pathway for enhancing the systemic resistance in crop plants.This suggests the importance of the transcription elements in activating the defense system during host-pathogen interactions.Our results showed that many genes in potato leaves up-regulated after P. infestans infection.This finding provides an overview of the underlying mechanisms related to the modulation and regulation of pathways in response to Phytophthora interactions.Consistent with this, previous studies manifested the induced expression of the responsive genes in the late blight resistance associated TFs such as WRKY, ERF, MAPK, bHLH Myc-type TF and NBS-LRR family genes 10 .We also observed differential regulation of MYB, MYB139, AP2-EREBP, C2H2L, and CC-LRR-NBS genes.Thus, our study highlights the role of TFs and other DNA binding proteins in defense response to P. infestans in potato.

Stress-resonsive genes, protein kinases and phytohormones control late blight resistance
Stress-responsive genes play an important role in imparting disease resistance in potato.Some of the stressresponsive genes and kinases in resistant genotypes were cytochrome P450 hydroxylase, serine-threonine protein kinase plant-type, and leucine-rich repeat receptor kinase.The role of cytochrome P450 has been deciphered for enhancing plant resistance via jasmonic acid and ethylene signaling pathways in soybean.Duan and co-workers 12 identified up-regulation of lignin-forming anionic peroxidase genes that may participate in ethylene-induced defense response against P. infestans.The protein kinases mainly calcium-dependent protein kinases (CDPKs) and leucine-rich repeat receptor-like protein kinases (LRR-RKs) determines key functions in pathogen recognition in lentil 23 .In the line of earlier research findings [10][11][12] , our results confirmed the involvement of signal transduction and stress-responsive genes in activating and maintaining a defense response to P. infestans in potato.The activation of signaling pathways is mostly regulated by salicylic acid, jasmonic acid, ethylene, which induce gene expression and defense response genes against P. infestans inoculation in plants 24 .We observed phytohormones related genes in resistant genotypes such as gibberellin regulated protein, BRASSINOSTER-OID INSENSITIVE 1-associated receptor kinase 1, ethylene-responsive proteinase inhibitor 1, and ethyleneresponsive transcription factor.Our findings were suppoted by Yang et al. 9 indicating gene expression profiling under exogenous ethylene application in late blight resistant potato genotype SD20.Consequently, they identified multiple signaling pathways including ethylene, salicylic acid, jasmonic acid, abscisic acid, auxin, cytokinin and gibberellin involved in SD20.It has been proven that ethylene-induced gene expression profiling provides insights into the ethylene signaling transduction pathway and its potential mechanisms in disease defense systems in potato.Moreover, the role of photosynthesis is well-known in plant growth and development.Researchers have described that most of the genes associated with photosynthesis pathways were down-regulated upon P. infestans inoculation leading to hypersensitive response and leaf lesion 25 .Thus, the stress-responsive genes, protein kinases and phytohormones do play crucial roles in conferring late blight resistance in potato.

Conclusion
Our study provides a landscape of transcriptome profiling in potato of diverse genetic backgrounds including interspecific somatic hybrid and common potato varieties.This illuminated the role of disease resistance, TFs, stress-responsive genes, and phytohormones genes imparting late blight resistance upon P. infestans infection under controlle conditions.We showed that key regulators of late blight resistance are disease resistance

Figure 2 .
Figure 2. Heat maps of top 50 differentially expressed genes (p < 0.05) for late blight resistance in potato somatic hybrid P7 versus Kufri Bahar (KB, control) by RNA-seq.In heat map, each horizontal line refers to a gene.Relatively up-regulated genes are shown in red colour, whereas down-regulated genes are shown in green colour.

Figure 3 .
Figure 3. Heat maps of top 50 differentially expressed genes (p < 0.05) in potato variety Kufri Girdhari (KG) versus Kufri Bahar (KB, control) by RNA-seq after artificial inoculation of P. infestans under controlled conditions.In heat map, each horizontal line refers to a gene.Relatively up-regulated genes are shown in red colour, whereas down-regulated genes are shown in green colour.

Figure 5 .
Figure 5. Gene Ontology (GO) characterization for cellular component, molceular fucntion, and biological process of up-regulated and down-regulated DEGs in P7 and Kufri Girdhari.

Figure 7 .
Figure 7.A cluster analysis based on the Neighbor-Joining method derived from the bootstrap consensus tree inferred from 100 replicates using the MEGA software showing the relationship among the 38 amino acid sequence of 18 genes involved in conferring late blight resistance in potato genotypes.

Table 1 .
Late blight resistant test of potato somatic hybrids and varieties by artificial inoculation of P. infestans under controlled conditions.

Table 3 .
Selected 2 H 2 -type zinc finger differentially expressed genes (p < 0.05) in the leaf tissues of potato genotypes after artificial inoculation with P. infestans by RNA-seq analysis.*Gene expression analysis in P7, Crd6, Kufri Girdhari and Kufri Jyoti was performed in comparison with susceptible control Kufri Bahar and expressed in term of Log 2 fold change.